Pharmaceutical composition having thrombolytic anti-inflammatory and cytoprotective properties

ABSTRACT

A pharmaceutical composition having thrombolytic, anti-inflammatory and cytoprotective properties is described. The composition contains active proteases attached to a gel or to a mixture of a gel with a water-soluble polymer, said attachment being achieved with the help of radiation, preferably with the help of gamma-radiation or a flow of accelerated electrons. As the proteases the composition contains proteases which are stable at a temperature of up to 70° C., preferably at 30-40° C., for instance such as subtilysine, trypsin, chemotrypsin, papain or streptokinase. The gel is preferably polyethylene glycol gel, dextran gel, or polyglycane gel, and the water-soluble polymer is polyethylene glycol, polyvinyl alcohol, dextran or polyglycane. The method of preparing said composition and its use are also disclosed. Advantages: the claimed composition is noted for a broad range of application and can be used in cardiology, nephrology, surgery, rheumatology, gynecology and gastroenterology.

FIELD OF THE ART

The present invention relates to medicine, particularly to pharmacologyand drugs based on enzyme preparations, and can be used in complextherapy for treating ischemic heart disease, ischemic cerebral strokes,rheumatoid processes and other diseases accompanied by ischemic,thrombotic, and non-specific inflammation phenomena.

This invention encompasses the field of enzymatic hydrolysis ofthrombogenic proteins, mainly fibrin, structure-forming protein of allthrombi, irrespective of the location and size of peptides. Moreparticularly, the invention relates to compounds containing proteolyticenzymes possessing fibrinolytic properties, immobilized on (attached to)hydrophilic water-soluble polymers. The invention is also associatedwith methods of using such compounds for fibrinolytic therapy intreating acute myocardial infarctions, ischemic cerebral strokes, andalso with therapeutic methods directed to the correction (improvement)of hemorheological characteristics in treating ischemic injuries oforgans and tissues of different etiology, including microthrombosis.

STATE OF THE ART

Known in the art are various medicinal preparations used for treatingischemic heart disease and ischemic cerebral strokes. As a rule, at thefinal stage of these diseases the main pathogenetic factor isintravascular thrombosis. In present-day medical practice thrombolyticmedicinal preparations have become widely used as etiotropic therapymeans for treating acute myocardial infarctions and ischemic cerebralstrokes. There are known pharmaceutical preparations, such as streptase,streptokinase, tissue plasminogen activator (alteplase) andfibrinolysine (Methodological Recommendations for Carrying out EarlyTherapeutic Measures in Patients with Acute Myocardial Infarction,Communication of the American Cardiologic College and American HeartAssociation. V. N. Ganyukov (Ed.), Collection of Papers, Novosibirsk,1998, p. 100 (Russian translation)). All these preparations, directly oras a result of activation of the anticoagulative system of blood, act onfibrin, leading to its destruction and, correspondingly, to lysis of theintravascular thrombus. In spite of their high therapeuticeffectiveness, these preparations have a pronounced side effect, namely,they can induce uncontrollable and dangerous hemorrhages, due todepletion of the coagulative system of blood (Saunders W. B. Indicationsfor Fibrinolytic Therapy Trialists Collaborative Group.//Lancet Ltd.,1994, vol. 343, pp. 311-322). Besides, for such preparations asstreptokinase and alteplase it is difficult to select an adequatetherapeutic dosage, because of the existence of an individual antiserumactivity to streptococcus in the human organism, that leads to theinactivation of these preparations.

Medicinal preparations are known, which are capable of diminishing theinflammatory reaction. The most widespread of these preparations arenon-steroidal anti-inflammatory preparations, such as aspirin,indomethacin, sodium diclofenac, and others (Nasonov E. L., Tsvetkova V.S., and Tov N. L., Selective inhibitors of cyclooxygenase-2: newprospects of treating human diseases//Ter. Arkhiv, 1998, No. 5, pp. 8-14(in Russian)). The known pharmaceutical preparations are not free fromessential disadvantages: they may cause stomach injuries withdevelopment of non-steroidal gastropathy, hemorrhages and ulcerations ofthe gastrointestinal tract mucosa.

Pharmaceutical preparations are known, which have cytoprotectiveproperties, such as preductal (cardioprotective effect) and blockers ofH₂ receptors (gastrozepine, ranitidine). The known preparations have aweak cytoprotective effect and prove to display pharmacological actionon prolonged administration (Brottier L., Barat J. L., Combie C. et al.,Therapeutic value of a cardioprotective agent in patients with severeischaemic cardiomyopathy//Eur. Heart J., 1990, vol. 11, pp. 207-212).

At present no data can be found in scientific and medical literatureconcerning the provision of pharmaceutical compositions producingmultipurpose synergic effect on the main pathogenetic links of ischemia,inflammation and thrombosis in combination with cytoprotectiveproperties.

Different methods for the immobilization of enzymes, includingproteases, on a number of polymeric carriers were reported. Forinstance, in the work by R. I. Salganik et al. “Immobilized ProteolyticEnzymes in Treating Purulent Processes”, Novosibirsk, pp. 3-8, 1981 (inRussian) a method is described for treating purulent wounds, abscessesand phlegmons with the help of proteolytic enzymes attached covalentlyto solid granules of cellulose. The use of liquid polymers with attachedproteases is described by O. A. Peretyagin et al. in “Oftalmologiya”,1987, No. 3, pp. 145-148 and by Gonchar et al. in “Veterinariya”, 1989,No. 4, pp. 52-55. In most cases a bifunctional chemical reagent was usedas the binding agent, in which one chemical group was linked to theamino acid residue of the enzyme protein, while another reactive groupwas linked to the polymeric carrier. A number of chemical procedures forthe covalent attachment of various proteins to solid carriers wereinvestigated. However, proteases attached to solid granules or fiberscannot be used for treating injuries or diseases which lead tointravascular thrombosis and are accompanied by negative changes ofhemorheology in the microcirculatory channel of tissues and organs.

Therefore, compounds are required, which effectively hydrolyze proteins,predominantly those responsible for thrombosis in blood vessels ofdifferent diameters, which constitute an etiological factor of thedevelopment of such diseases as acute myocardial infarction and ischemiccerebral stroke. At present such compounds containing nontoxicthrombolytics and substances with hemorheological corrective functionshave not been synthesized for complex therapy of ischemic heart disease,hypertensive disease and rheumatoid diseases. Simple, inexpensive andconvenient methods are also required for simultaneously attachingproteases to a complex of polymeric carriers and providing sterilizationof the end product. Ideal methods are those which can provide compoundshaving different viscosity and aggregate state, suitable for differentpurposes and different ways of administration.

ESSENCE OF THE INVENTION

The herein-presented investigation meets the above-said requirements andalso offers relative advantages over other medicinal preparations andmethods of preparing pharmaceutical compositions featuring a complexsynergic therapeutic effect on various links of the pathogenesis ofischemic heart disease. The presented investigation, the claimedpharmaceutical composition, and methods of preparing thereof ensure theobtaining of high therapeutic effect in treating non-specificinflammatory processes owing to cytoprotective, thrombolytic andanti-ischemic effects.

The proposed invention is directed to the provision of compositions forselective hydrolysis of thrombogenic proteins, consisting of activeproteases attached to a combination of hydrophilic water-solublepolymers. Said compositions predominantly consist of subtilysinesimmobilized on hydrophilic polymers.

This invention contributes to the development of methods of making suchcompositions by attaching proteases to water-soluble polymers to producea mixture and simultaneously sterilize said mixture by irradiation,particularly with the help of a stream of accelerated electrons orgamma-radiation, as well as other kinds of ionizing radiation, includinglaser radiation sources.

The proposed invention is directed to novel pharmaceutical compositionsfor the hydrolysis of thrombogenic proteins, consisting of activeproteases attached to hydrophilic polymers. Such compositions are usefulfor the removal of thrombi comprising fibrin and other formations. Theproposed invention is also directed to novel compounds for correctingpathological changes in tissues and organs, originated as a result ofthrombosis and ischemia, as well as to the correction of non-specificinflammatory reactions, and as an auxiliary means for treating andpreventing gastropathies developing as a result of administeringnon-steroidal anti-inflammatory preparations. Proteases immobilized onhydrophilic polymers according to the invention selectively degradethrombogenic proteins, mainly fibrin, while live cells and functionallyactive proteins remain intact and undamaged. For effective hydrolysis ofthe proteins of thrombotic masses, it is preferable to have apolyfunctional mixture of proteases which are capable of recognizing andhydrolyzing various peptide linkages. Natural proteases include, forinstance, subtilysine, trypsin, chemotrypsin, papain and other enzymesof animal and bacterial origin, having fibrinolytic and proteolyticactivity.

Though proteases are obtainable from any sources, including animal andvegetable ones, bacterial proteases are especially preferable. Comparedwith other types of proteases, bacterial proteases are usually lesscostly and are available in unlimited amounts. Preferable bacterialproteases of the proposed invention can be reproduced. by Bac. subtilisand are known as subtilysines. A mixture of neutral and/or alkalinesubtilysines is preferable, because the mixture can selectively degradea variety of peptide linkages and preserve activity at pH from 6.0 to10.0.

The proteases according to the proposed invention are attached to acombination of water-soluble polymers which act as carriers forproteolytic enzymes. Preferable water-soluble polymers are polyethyleneoxide (PEO), synonym: polyethylene glycol (PEG), having a molecularweight of 1500 kDa (PEO-1500), and dextran having a molecular weight of30-40 kDa. Polyethylene oxides and dextrans having a smaller or greatermolecular weight can also be used instead of or together with PEO-1500and dextran having a molecular weight of 30-40 kDa. Other suitablewater-soluble polymers and their combinations that also can be usedinclude, e.g., polyvinyl alcohol, polyvinylpyrrolidone, and the like.

The compositions according to the proposed invention can be in anynon-solid or solid form or in a combination of aggregate forms. It ispreferable to use lyophilized powders (substances) in variouscombinations with water-soluble polymers as the compositions, dependingon the field in which the compound is planned to be used.

For the provision of the overall therapeutic properties, antibodies,therapeutic disinfectants, glucocorticoids, tissue regenerationstimulants, and other therapeutic agents can be added to the givencomposition. To the benefit of the case in hand, such agents should notbe mixed in a considerable amount with proteases, unless their effect onthe specific and therapeutic activity of the claimed pharmaceuticalcomposition has been circumstantiated (investigated).

The present invention is also directed to methods of preparingcompositions by attaching proteases to water-soluble polymers andsimultaneously sterilizing the mixture by radiation. Preferable kinds ofradiation are accelerated electrons or gamma-radiation.

Briefly, the technology is as follows: a solution of a mixture ofproteases, preferably subtilysines, and water-soluble polymers,preferably polyethylene oxide and dextran, is subjected to the action ofelectrons emitted by an electron accelerator in a dose which promotesattaching thereof to a polymeric carrier and simultaneously sterilizingthe obtained compound. On the other hand, gamma-rays emitted by Co⁶⁰ canbe used instead of an electron bunch with the provision of attaching andsterilizing the components. A sufficient radiation dose can be selectedby a person skilled in the art by calculating the conditions for theprovision of the mixture sterility and inessential effect on theproteolytic activity of the enzymes.

Finally, the proposed invention is also directed to methods of cleaningthe vascular bed from thrombi by controlling or using effective amountsof the claimed composition for the removal of this material. Thecompositions according to the present invention are effective when usedas preparations for pharmaceutical purposes in different ways andfields, such as medicine, stomatology, veterinary medicine, and personalcare.

In this connection, the compositions according to the present inventionare particularly eligible for treating ischemic heart disease and itscomplications, as well as ischemic cerebral strokes, rheumatoid diseasesand other pathologies, in whose pathogenesis there take place aninflammatory reaction, ischemia of tissues, disturbances of hemorheologyand vascular microcirculation because of thrombosis. The claimedpharmaceutical composition is also suitable for enzymatic destruction ofviscous biological fluids, such as secreted mucosae (e.g., bronchialsecret), therefore it is effective in treating pulmonary diseases forthe prevention and treatment of bronchial obstruction. The claimedpharmaceutical composition is also applicable for the enzymaticdestruction of proteins and peptides of necrotic tissues which arealways present in traumatic and infectious-inflammatory processes.Therefore this composition is also effective in treating pyoinflammatorydiseases of different etiology and localization.

These compositions can be applied to the focus of injury by any methodknown in the medicine. Such methods include, for instance, parenteraladministration, intracavitary and external use, aerosol spraying,syringing through a trocar, catheters, bronchoscope, or other suitableuse. The method of administration will depend, at least partially, onthe nature of the injured. area and on the type and quantity ofthrombogenic or necrotic tissues to be removed. The predominant methodof administration is parenteral administration of the preparation(compositions).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the thrombolytic properties of the claimedcomposition, investigated on a model of thrombolysis in vitro.

The claimed composition in the standard therapeutic dose of 50physiological units/ml (50 PhU/ml) reliably excels fibrinolysine(p<0.02); trypsin (p<0.01); and spontaneous thrombolysis inphysiological solution (p<0.01).

FIG. 2 demonstrates that the thrombolytic properties of the claimedcomposition are preserved as the thrombus “age” increases to 7 days.Fibrolysine does not act on the 7-days thrombus. During the first twohours the action of fibrolysine does not differ with certainty from thespontaneous background lysis in physiological solution. By the end ofthe 4th hour the claimed composition dissolves the thrombus completely,while fibrinolysine lyses only 20% of the mass of the “old” thrombus.

FIG. 3 demonstrates the anti-inflammatory properties of he claimedcomposition, investigated on a model simulating induction of aninflammation mediator-tumor necrosis factor (αTNF) in CBA-line mice withendotoxic shock. The activity of the αTNF was measured using L929 linecells and was expressed in activity units (AU):

1—αTNF activity in intact animals;

2—αTNF activity in animals after administration of endotoxin;

3—αTNF activity in animals to which the claimed composition wasadministered one hour before the administration of endotoxin. As isseen, the claimed composition reduces 50-fold the αTNF activity.

FIG. 4 demonstrates the cytoprotective properties of the claimedcomposition, studied on a model of adrenaline myocarditis in rats. Dataof histological morphometric investigations of the volume of necrosesand dystrophic changes in the myocardium of the rats are presented. Thevolume of injuries was determined planimetrically and expressed inpercent of the volume density which is equal to the volume ofinjuries/total value of tissue ratio multiplied by 100%.

K necr.—control animals to which after administering adrenaline 1 ml ofisotonic NaCl solution was administered intraperitoneally twice a day.

TRB necr.—experimental animals to which after administering adrenaline 1ml of the claimed composition was administered intraperitoneally twice aday.

The first two columns demonstrate positive effect of the claimedcomposition (approximately 1.5-fold reduction of the number of necrosesin the cardiac muscle) by the end of the third day of treating theanimals.

The next two columns demonstrate positive effect of the claimedcomposition (approximately 2.5-fold reduction of the number ofdystrophic changes in the cardiac muscle) by the end of the seventh dayof treating the animals.

The Examples which follow are intended to illustrate the formulation,the method of preparing and the pharmacological properties of theclaimed composition, but not to limit the proposed invention.

PREFERRED EMBODIMENTS OF THE INVENTION Example 1

The proposed pharmaceutical composition is prepared in the followingmanner:

a reaction mixture is prepared by dissolving protosubtilin,predominantly protosubtilin G3Kh and polyethylene oxide (PEO) having amolecular weight of 400-20000 Da (predominantly 1500 Da) in a solutionof dextran having a molecular weight of 40-70 kDa (predominantly 40 kDa)in a 0.025 M sodium phosphate buffer solution with pH 7.5-8.2. Theobtained mixture is purified by removing ballast proteins by saltprecipitation and subsequent filtration. The resulting solution issubjected to irradiation with gamma-rays or a stream of acceleratedelectrons (with energy of 2.0 MeV) in a dose of 0.5-1.5 Mrad. Thesolution after irradiation is subjected to sterilizing filtration andpackaged in 10 ml batches into 15 ml vials. Then the solution islyophilized to a residual moisture content not exceeding 2%.

As a result, a composition is obtained, containing a protease complexfrom Bac. subtilis, immobilized on polyethylene oxide and dextran. Theproteolytic activity of the obtained composition in one vial is from 500to 1000.0 proteolytic units per gram (PU/g). The composition comprises aslightly yellowish porous homogeneous mass.

For therapeutic purposes a solution of the claimed composition is used(conditional appellation thereof being “Trombovazim”), which is preparedex tempore by dissolving the contents of a vial in 10 ml of sterileisotonic solution of sodium chloride or in 10 ml of water forinjections.

Example 2

The claimed composition can be prepared by another method, namely, bypreparing separately an active component (component (1)) which containsa complex of proteases immobilized on polyethylene oxide and dextran,and a solvent which is a solution of polyethylene oxide (component (2)).The two-component formulation of the composition makes it possible tovary the activity of the claimed pharmaceutical composition by varyingthe active component/solvent ratio, so that individual schemes oftreating can be selected. The two-component composition is prepared inthe following manner:

Component (1) (Active Substance)

The reaction mixture is prepared by dissolving protosubtilin(predominantly protosubtilin G3Kh) and polyethylene oxide (PEO) having amolecular weight of 400-20000 Da (predominantly 1500 Da) in a solutionof dextran having a molecular weight of 70-40 kDa (predominantly 40 kDa)in a 0.025 M sodium phosphate buffer solution with pH 7.5-8.2. Theobtained mixture is purified by removing ballast proteins by saltprecipitation and subsequent filtration. The resulting solution issubjected to irradiation with gamma-rays or a stream of acceleratedelectrons (with energy of 2.0 MeV) in a dose of 1.0 Mrad. The solutionafter irradiation is subjected to sterilizing filtration and packaged in10 ml batches into 15 ml vials. Then the solution is lyophilized to aresidual moisture content not exceeding 2%.

As a result, component (1) of the composition is obtained, whichcomponent contains a protease complex from Bac. subtilis, immobilized onpolyethylene and dextran. The proteolytic activity of the obtainedcomposition is from 500 to 1000.0 PU/g. Component (1) comprises a porousslightly yellowish homogeneous mass.

Component (2) (Solvent)

A solution of polyethylene oxide (predominantly polyethylene oxide witha molecular weight of 1500 Da) is prepared in a 0.025 M sodium phosphatebuffer solution with pH 7.5-8.2. The obtained solution is subjected toirradiation with gamma-rays or a stream of accelerated electrons (withenergy of 2.0 MeV) in a dose of 1.0 Mrad. The solution after irradiationis subjected to sterilizing filtration and packaged in 10 ml batchesinto 15 ml vials.

As a result, a sterile solvent for component (1) of the claimedcomposition is obtained. The solvent comprises a slightly yellowishliquid.

For therapeutic purposes a solution of the claimed composition is used(conditional appellation thereof being “Trombovazim”), which is preparedex tempore by dissolving the contents of a vial with component (1)containing immobilized proteases in 10 ml of component (2) (solvent).

Dissolution ex tempore of component (1) in the solvent (component (2))gives the claimed pharmaceutical composition.

Example 3

15 of polyethylene oxide PEO-1500 are dissolved in 300 ml of a 10%solution of dextran having a molecular weight of 40 kDa in a 0.025 Msodium phosphate buffer with pH 7.5, 6.3 g of protosubtilin G3Kh areadded, the mixture is stirred at a temperature of 18-20° C. for 30minutes. Then ballast proteins are precipitated by salt precipitationtechniques. For this to be done, added to the mixture in succession tillcomplete dissolution are 1.3 g of sodium phosphate disubstituted tofinal concentration of 0.45% and 1.9 g of calcium chloride to finalconcentration of 0.63%. After the dissolution of calcium chloride aninsoluble precipitate of calcium phosphate is formed in the reactionmixture, and this calcium phosphate adsorbs the ballast proteins. Themixture is maintained for 12 hours at a temperature of 4 to 8° C. forcomplete precipitation of the ballast proteins. After that the reactionmixture is filtered through paper filters (“white ribbon”). The volumeof the filtrate is 300 ml. The obtained solution is subjected to gammairradiation in a dose of 1.0 Mrad. After the irradiation the solution issubjected to sterilizing filtration, packaged in batches of 10 ml in 15ml vials and lyophilized to a residual moisture content not exceeding2%. As a result, a composition is obtained, having the followingformulation in weight percent: 1. Protosubtilin G3Kh 2.1 2. Dextran(molecular weight 40 kDa) 10.0 3. Polyethylene oxide PEO-1500 5.0 4.0.025 M sodium phosphate buffer 82.9

The proteolytic activity of the composition is 850 PU/g.

Example 4

15 of polyethylene oxide PEO-1500 are dissolved in 300 ml of a 5%solution of dextran having a molecular weight of 40 kDa in a 0.025 Msodium phosphate buffer with pH 8.2, 6.0 g of protosubtilin G3Kh areadded, the mixture is stirred at a temperature of 18-20° C. for 30minutes. Then ballast proteins are precipitated by salt precipitationtechniques. For this to be done, added to the mixture in succession tillcomplete dissolution are 1.3 g of sodium phosphate disubstituted tofinal concentration of 0.45% and 1.9 g of calcium chloride to finalconcentration of 0.63%. After the dissolution of calcium chloride aninsoluble precipitate of calcium phosphate is formed in the reactionmixture, and this calcium phosphate adsorbs the ballast proteins. Themixture is maintained for 12 hours at a temperature of 4 to 8° C. forcomplete precipitation of the ballast proteins. After that the reactionmixture is filtered through paper filters (“white ribbon”). The volumeof the filtrate is 300 ml. The obtained solution is subjected to gammairradiation in a dose of 1.0 Mrad. After the irradiation the solution issubjected to sterilizing filtration, packaged in batches of 10 ml in 15ml vials and lyophilized to a. residual moisture content not exceeding2%. As a result, a composition is obtained, having the followingformulation in weight percent: 1. Protosubtilin G3Kh 2.0 2. Dextran(molecular weight 40 kDa) 5.0 3. Polyethylene oxide PEO-1500 5.0 4.0.025 M sodium phosphate buffer 88.0

Example 5

0.5 of polyethylene oxide PEO-1500 is dissolved in 300 ml of a 10%solution of dextran having a molecular weight of 40 kDa in a 0.025 Msodium phosphate buffer with pH 8.2, 5.7 g of protosubtilin G3Kh areadded, the mixture is stirred at a temperature of 18-20° C. for 30minutes. Then ballast proteins are precipitated by salt precipitationtechniques. For this to be done, added to the mixture in succession tillcomplete dissolution are 1.3 g of sodium phosphate disubstituted tofinal concentration of 0.45% and 1.9 g of calcium chloride to finalconcentration of 0.63%. After the dissolution of calcium chloride aninsoluble precipitate of calcium phosphate is formed in the reactionmixture, and this calcium phosphate adsorbs the ballast proteins. Themixture is maintained for 12 hours at a temperature of 4 to 8° C. forcomplete precipitation of the ballast proteins. After that the reactionmixture is filtered through paper filters (“white ribbon”). The volumeof the filtrate is 300 ml. The obtained solution is subjected to gammairradiation in a dose of 1.0 Mrad. After the irradiation the solution issubjected to sterilizing filtration, packaged in batches of 10 ml in 15ml vials and lyophilized to a residual moisture content not exceeding2%. As a result, a composition is obtained, having the followingformulation in weight percent: 1. Protosubtilin G3Kh 1.9 2. Dextran(molecular weight 40 kDa) 10.0 3. Polyethylene oxide PEO-1500 0.5 4.0.025 M sodium phosphate buffer 87.6

The proteolytic activity of the composition is 750 PU/g.

Example 6

15 of polyethylene oxide PEO-1500 are dissolved in 300 ml of a 5%solution of dextran having a molecular weight of 40 kDa in a 0.025 Msodium phosphate buffer with pH 8.2, 7.5 g of protosubtilin G3Kh areadded, the mixture is stirred at a temperature of 18-20° C. for 30minutes. Then ballast proteins are precipitated by salt precipitationtechniques. For this to be done, added to the mixture in succession tillcomplete dissolution are 1.3 g of sodium phosphate disubstituted tofinal concentration of 0.45% and 1.9 g of calcium chloride to finalconcentration of 0.63%. After the dissolution of calcium chloride aninsoluble precipitate of calcium phosphate is formed in the reactionmixture, and this calcium phosphate adsorbs the ballast proteins. Themixture is maintained for 12 hours at a temperature of 4 to 8° C. forcomplete precipitation of the ballast proteins. After that the reactionmixture is filtered through paper filters (“white ribbon”). The volumeof the filtrate is 300 ml. The obtained solution is subjected to gammairradiation in a dose of 1.2 Mrad. After the irradiation the solution issubjected to sterilizing filtration, packaged in batches of 10 ml in 15ml vials and lyophilized to a residual moisture content not exceeding2%. As a result, a composition is obtained, having the followingformulation in weight percent: 1. Protosubtilin G3Kh 2.5 2. Dextran(molecular weight 40 kDa) 5.0 3. Polyethylene oxide PEO-1500 5.0 4.0.025 M sodium phosphate buffer 87.5

The proteolytic activity of the composition is 500 PU/g.

Example 7

15 of polyethylene oxide PEO-4000 are dissolved in 300 ml of a 5%solution of dextran having a molecular weight of 70 kDa in a 0.025 Msodium phosphate buffer with pH 8.2, 7.5 g of protosubtilin G10Kh areadded, the mixture is stirred at a temperature of 18-20° C. for 30minutes. Then ballast proteins are precipitated by salt precipitationtechniques. For this to be done, added to the mixture in succession tillcomplete dissolution are 1.3 g of sodium phosphate disubstituted tofinal concentration of 0.45% and 1.9 g of calcium chloride to finalconcentration of 0.63%. After the dissolution of calcium chloride aninsoluble precipitate of calcium phosphate is formed in the reactionmixture, and this calcium phosphate adsorbs the ballast proteins. Themixture is maintained for 12 hours at a temperature of 4 to 8° C. forcomplete precipitation of the ballast proteins. After that the reactionmixture is filtered through paper filters (“white ribbon”). The volumeof the filtrate is 300 ml. The obtained solution is subjected to gammairradiation in a dose of 0.8 Mrad. After the irradiation the solution issubjected to sterilizing filtration, packaged in batches of 10 ml in 15ml vials and lyophilized to a residual moisture content not exceeding2%. As a result, a composition is obtained, having the followingformulation in weight percent: 1. Protosubtilin G10Kh 2.5 2. Dextran(molecular weight 70 kDa) 5.0 3. Polyethylene oxide PEO-4000 5.0 4.0.025 M sodium phosphate buffer 87.5

The proteolytic activity of the composition is 1000 PU/g.

Example 8 Preparing the Claimed Composition Consisting of an ActiveComponent and a Solvent (Two-Component Formulation)

Component (1):

0.5 of polyethylene oxide PEO-1500 are dissolved in 300 ml of a 10%solution of dextran having a molecular weight of 40 kDa in a 0.025 Msodium phosphate buffer with pH 8.2, 5.7 g of protosubtilin G3Kh areadded, the mixture is stirred at a temperature of 18-20° C. for 30minutes. Then ballast proteins are precipitated by salt precipitationtechniques. For this to be done, added to the mixture in succession tillcomplete dissolution are 1.3 g of sodium phosphate disubstituted tofinal concentration of 0.45% and 1.9 g of calcium chloride to finalconcentration of 0.63%. After the dissolution of calcium chloride aninsoluble precipitate of calcium phosphate is formed in the reactionmixture, and this calcium phosphate adsorbs the ballast proteins. Themixture is maintained for 12 hours at a temperature of 4 to 8° C. forcomplete precipitation of the ballast proteins. After that the reactionmixture is filtered through paper filters (“white ribbon”). The volumeof the filtrate is 300 ml. The obtained solution is subjected to gammairradiation in a dose of 1.0 Mrad. After the irradiation the solution issubjected to sterilizing filtration, packaged in batches of 10 ml in 15ml vials and lyophilized to a residual moisture content not exceeding2%. As a result, component (1) of the composition is obtained, havingthe following formulation in weight percent: 1. Protosubtilin G3Kh 1.92. Dextran (molecular weight 40 kDa) 10.0 3. Polyethylene oxide PEO-15000.5 4. 0.025 M sodium phosphate buffer 87.6

The proteolytic activity of component (1) is 750 PU/g.

Component (2) (Solvent):

15.0 g of polyethylene oxide PEO-1500 are dissolved in 300 ml of a 0.025M sodium phosphate buffer. Then the solution is filtered through paperfilters (“white ribbon”). The volume of the filtrate is 300 ml. Theobtained solution is subjected to gamma irradiation in a dose of 1.0Mrad. After the irradiation the solution is subjected to sterilizingfiltration and packaged in batches of 10 ml in 15 ml vials. As a result,component (2) of the composition is obtained, having the followingformulation in weight percent: 1. Polyethylene oxide PEO-1500 5.0 2.0.025 M sodium phosphate buffer 95.0

The dissolution of component (1) in the solvent (component (2)) andlyophilization give the claimed pharmaceutical composition.

Example 9 Investigation of the Pharmacological Properties of the ClaimedComposition (“Trombovazim”)

The pharmacological properties of the claimed composition (itsconditional appellation being “Trombovazim”) have been checked underlaboratory conditions in vitro and in vivo.

The thrombolytic properties of the composition have been investigated ona model of thrombolysis in vitro (FIG. 1). As can be seen from FIG. 1,“Trombovazim” produces a sharply pronounced thrombolytic effect,reliably exceeding in the standard therapeutic concentration of 50PhU/ml the effect of fibrinolysine (p<0.02); trypsin (p<0.01); andspontaneous thrombolysis in physiological solution (p<0.01). It shouldbe noted that as the “age” of the thrombus increases to 7 days, thethrombolytic properties of “Trombovazim” are preserved (FIG. 2), whilefibrinolysine practically does not act on the 7-days thrombus. Duringthe first two hours the action of fibrolysine does not differ withcertainty from the spontaneous background lysis in physiologicalsolution. By the end of the 4th hour the claimed composition dissolvesthe thrombus completely, while fibrinolysine lyses only 20% of the massof the “old” thrombus.

It is known that fibrinolysine is the most active fibrinolytic (2),while such preparations as streptokinase, urokinase, alteplase andtissue plasminogen activator are indirect fibrinolytics and theirthrombolytic action is mediated by the activation of the fibrinolysissystem and production of endogenous fibrinolysine, the latter leading tothe lysis of the formed thrombus (1).

The specific thrombolytic activity of “Trombovazim” in vivo has beeninvestigated on a model of carotid artery thrombosis induced in rats bythe application of ferrous chloride.

The results of the effect of prophylactic administering “Trombovazim”(80 PU per animal) on the blood flow (ml/min) along ipsilateral carotidarteries after the application of ferrous chloride (0-15 min) in Wistarline rats are presented in Table 2. TABLE 2 Group 0 min 15 min 30 min 45min 60 min 75 min 90 min Control n = 9 3.8 ± 0.3 2.3 ± 0.4 0.9 ± 0.5 0.4± 0.2 0.5 ± 0.4 0.2 ± 0.1 0.2 ± 0.1 Experiment n = 10 2.9 ± 0.3 2.5 ±0.1 2.1 ± 0.3 1.9 ± 0.4 1.9 ± 0.5 1.9 ± 0.4 1.7 ± 0.4

As is seen from the presented results, prophylactic administration of“Trombovazim” effectively limits the process of thrombosis in thecarotid artery during the first 1.5 hours after the effect of ferrouschloride thereon.

The results of the effect of therapeutic administering “Trombovazim” (80PU per animal) on the blood flow (ml/min) along ipsilateral carotidarteries after the application of ferrous chloride (0-15 min) in Wistarline rats are presented in Table 3. TABLE 3 Group 0 min 15 min 30 min 45min 60 min 75 min 90 min Control n = 8 3.3 ± 0.4 2.6 + 0.6 2.8 ± 0.9 2.1± 0.7 2.3 + 0.7 1.2 ± 0.4 0.9 ± 0.4 Experiment n = 9 3.8 ± 0.3 3.3 ± 0.52.7 + 0.5 1.7 ± 0.4 1.6 ± 0.3 1.6 + 0.4 1.8 ± 0.4

As is seen from the presented results, therapeutic administration of“Trombovazim” effectively inhibits the process of thrombosis in thecarotid artery during the first 1.5 hours after the effect of ferrouschloride thereon. Besides, histological (morphometric) investigationshave shown that upon therapeutic administration of “Trombovazim” after24 hours the proportion of animals with complete occlusion of thecarotid artery and with the presence of a thrombus in it reduces 3-fold,this being indicative of a pronounced thrombolytic activity of thepreparation.

The anti-inflammatory properties of the claimed composition have beeninvestigated on a model of inducing one of the main inflammationmediators-tumor necrosis factor (αTNF) in mice of the CBA line withendotoxic shock (FIG. 3). The activity of the αTNF was measuredbiologically, using L929 line cells and was expressed in units of action(UA). Shown in FIG. 3 are: the αTNF activity in intact animals (1) andin animals after endotoxin administration (2). The administration of“Trombovazim” one hour before the administration of endotoxin (3)reduces 50-fold the αTNF activity, this proving the anti-inflammatoryproperties of “Trombovazim”.

The cytoprotective properties of “Trombovazim” have been studied on amodel of indomethacin gastropathy (Table 1) and adrenaline myocarditisin rats (FIG. 4). TABLE 1 Experiment: 6.2 mg of indomethacin Control:6.2 mg of indomethacin administered administered intragastrically 1 hourintragastrically 1 hour before experimental before experimental effectof intra- effect of intraperitoneal administration peritonealadministration of 3 ml of No. of of 3 ml of physiological solution“Trombovazim” animal S_(stomach), S_(bleeding), % of injuries,S_(stomach), S_(bleeding), % of injuries, in group mm² SH mm² SB S% mm²SH mm² SB S% 1 910 17.3 1.9 830 29.2 3.5 2 685 26.9 3.9 778 13.6 1.8 3650 24.0 3.7 845 12.2 1.4 4 784 18.4 2.3 745 8.54 1.1 5 726 34.3 4.7 8237.6 1.1 6 927 22.7 2.5 769 0 0 7 699 46.6 6.7 778 0 0 8 853 17.0 2.0 57510.2 1.8 9 892 34.1 3.8 653 16.2 2.4 10 — — — 568 0 0 X ± SD 792 + 10626.8 ± 9.9 3.5 ± 1.5 715 ± 151 9.8 ± 9.0 1.3 + 1.2

From the presented Table it follows that the control and experimentalgroups of the rats are comparable in terms of the total stomach area(SH): there was no reliable difference between the characteristics. Thearea of bleedings (SB) in the groups reliably differed: in the groupwith preliminary administration of “Trombovazim” (experiment) SB is 3times smaller than in control (p<0.01). The same reliable relationshipis observed when comparing the relative affected area (S %). Hence,“Trombovazim” displays a pronounced cytoprotective effect in the case ofindomethacin injury of the stomach mucosa.

FIG. 4 shows the data of histological morphometric investigation of thevolume of necroses and dystrophic changes in the myocardium of rats withadrenaline myocarditis. The volume of injuries was determinedplanimetrically and expressed in percent of the volume density which isequal to the volume of injuries/total value of tissue ratio multipliedby 100%. In the control group (K), after administering adrenaline, 1.0ml of isotonic NaCl solution was administered intraperitoneally twice aday; in the experimental group (TRB) 1 ml of “Trombovazim” wasadministered intraperitoneally twice a day. From the presented resultsit follows that after the development of adrenaline myocarditis thetreatment with “Trombovazim” reliably reduces 1.5-fold the number ofnecroses in the cardiac muscle by the end of the 3rd day: (TBR necr.)compared with control K necr.). Dystrophic changes in the cardiac musclein the experimental group (TBR dystr.) by the end of the 7th day arereliably 2.5 times smaller than in the control group (K dystr.).

The results presented in Table 1 and in FIG. 4 prove that “Trombovazim”has pronounced cardioprotective and cytoprotective activity. Theprotective effect of “Trombovazim” reliably manifests itself in specificgastropathy caused by non-steroidal anti-inflammatory preparations,particularly by indomethacin, and the therapeutic effect of“Trombovazim” reliably manifests itself in acute adrenaline myocarditis,in whose pathogenesis the key role is played by acute ischemia, necrosiswith the development of myocardial dystrophy.

The disintoxication, anti-ischemic and cytoprotective effects wereinvestigated also on a model of the liver ischemia/reperfusion. For thispurpose, in the experimental and control groups of Wistar line rats (10rats in each group) the liver triad in the animals after laparotomy wasclamped for 20 minutes, i.e., the regional lymph and blood circulationwere completely blocked. Then the blood flow was restored, and thehistological structure of the liver in the animals was investigated (themain quantitative criterion was the accumulation of leukocytes inregional lymphatic nodes as the characteristic of response to thecytotoxic injury of the liver tissue by the products ofischemia/reperfusion: peroxides and free radicals). The results of theinvestigation are presented in FIG. 5.

As is seen from the presented results, “Trombovazim” produces ahepatoprotective effect on the model of liver ischemia/reperfusion, andthis is an additional confirmation of the cytoprotective,disintoxication and anti-inflammatory properties of “Trombovazim”.

Though the description of the present invention is commendatory for thepreferable use thereof, it should be understood that variousmodifications may be made therein without departing from the true spiritof the present invention. Therefore, the invention is limited only bythe following set of claims.

1. A composition having thrombolytic, anti-inflammatory, disintoxicationand cytoprotective properties for treating diseases accompanied byischemia, thrombosis, intoxication, inflammation, said compositionconsisting of active proteases, immobilized on a mixture ofwater-soluble polymers.
 2. A composition according to claim 1,characterized in that immobilization of proteases from the mixture ofwater-soluble polymers is effected with the help of ionizing radiation,by acting therewith on an aqueous solution containing an active proteaseand a mixture of water-soluble polymers.
 3. A composition according toclaim 1, characterized in that the ionizing radiation is a flow ofaccelerated electrons, gamma-radiation and UV-radiation.
 4. Acomposition according to claim 1, characterized in that proteolyticenzymes obtained from microbiological, animal or vegetable startingmaterial are used as the active protease.
 5. A composition according toclaim 4, characterized in that proteolytic enzymes produced by Bacillussubtilis are used as the active protease.
 6. A composition according toclaim 1, characterized in that a mixture of polyethylene oxide anddextran is used as the mixture of water-soluble polymeric carriers forthe immobilization of active proteases.
 7. A composition according toclaim 1, characterized in that it comprises a one-component medicinalform containing lyophilized active proteases immobilized on a mixture ofpolyethylene oxide and dextran, and also combinations with aspirin,indomethacin and diclofenac.
 8. A composition according to claim 1,characterized in that it comprises a two-component medicinal formcontaining lyophilized active proteases immobilized on a mixture ofpolyethylene oxide with dextran and a solvent.
 9. A compositionaccording to claim 8, characterized in that a solution of polyethyleneoxide activated by ionizing radiation is used as the solvent.
 10. Acomposition according to claim 1, characterized in that it is aneffective therapeutic agent for treating: ischemic heart disease andacute myocardial infarctions, ischemic disturbances of cerebralcirculation, arterial and venous thromboses, obstructive diseases of thelungs, gastrites and stomach ulcer, gynecological, stomatological andsurgical diseases.
 11. A composition according to claim 10,characterized in that the therapeutic effect is attained uponparenteral, oral, intracavitary and local administration.
 12. Acomposition producing a protective effect on the stomach mucosa uponcombined administration with non-steroidal analgesics.
 13. A method ofpreparing a composition of claim 1, comprising simultaneous attachmentof proteases to a gel or to a mixture of a gel with a water-solublepolymer for the formation of a non-solid mixture, and radiationsterilization of the mixture.
 14. A method according to claim 13,wherein the radiation is a flow of accelerated electrons orgamma-radiation.
 15. A method according to claim 13, wherein subtilysineis used as said proteases, polyethylene glycol is used as said gel, andsaid water-soluble polymer is polyethylene glycol.
 16. A method ofprotein hydrolysis, consisting in applying an effective amount of acompound consisting of proteases attached to a gel or to a mixture of agel with a water-soluble polymer for the hydrolysis of theabove-mentioned proteins.
 17. A method according to claim 16, whereinthe compound is applied by way of local application, as an aerosol, byinjection, through a trocar, bronchoscopically or with the help of aturunda.
 18. A method according to claim 16, wherein said proteases aresubtilysine, chemotrypsin, papain or streptokinase.
 19. A methodaccording to claim 18, wherein said protease is subtilysine.
 20. Amethod according to claim 16, wherein said gel is a polyethylene glycolgel, dextran gel or polyglycane gel.
 21. A method according to claim 16,wherein said gel is a polyethylene glycol gel.
 22. A method according toclaim 16, wherein said water-soluble polymer is polyethylene glycol,polyvinyl alcohol, dextran or polyglycane.
 23. A method according toclaim 22, wherein said polymer is polyethylene glycol.